From the studies using receptor selective antagonists and receptor knock-out mice, it is apparent that the activation of delta-opioid receptor is intimately involved in the morphine tolerance development. Hence, the control of delta-opioid receptor activity will have a pronounced effect on the pharmacological actions of morphine. Prolonged activation of the delta-opioid receptor will lead to receptor desensitization and internalization. Additionally, a superactivation of adenylyl cyclase activity can be observed after the removal of agonist or in the presence of antagonist. Being a member of the superfamily of G protein-coupled receptors (GPCRs), these cellular adaptational responses stem from covalent modification of the receptor, such as phosphorylation. Indeed, delta-opioid receptor, similar to many GPCRs is phosphorylated in the presence of agonist. During the last funding period, using mutagenesis approach, we have identified the Ser/Thr residues that are phosphorylated. Agonist such as DPDPE caused a hierarchical phosphorylation of Ser[363] and Thr[358] in the carboxyl tail domain of the receptor. The blockade of the phosphorylation of these sites altered the rate of receptor internalization and desensitization, but did not abolish these two cellular events. Similarly, the superactivation of the adenylyl cyclase was attenuated but not abolished with the mutation of these Ser/Thr residues. Since the phosphorylation of GPCR has led to an increase in the affinity for the arrestin binding, and the arrestin could interact with agonist-activated GPCR without receptor phosphorylation, it is our hypothesis that the failure to observe the blockade of the receptor desensitization and internalization in the phosphorylation minus mutants is due to the ability of the arrestin to bind to these receptor mutants. Thus, in the current proposed studies, we will identify the amino acid residues in addition to those that are phosphorylated within the delta-opioid receptor that are involved in the arrestin binding. We will establish initially the protein kinases and the sites of phosphorylation in various cells, including primary DRG neuron cultures. The involvement of other receptor domains in addition to the carboxyl tail domain, such as the 2nd and 3rd intracellular loops, in the arrestin interaction will be determined by biochemical and surface plasmon resonance studies. Combining with mutational analyses of the receptor domains involved both in the GST-fusion protein format, or with the GFP-arrestin translocation measurements, we should be able to determine the binding pocket in the receptor for arrestin. The effect of such mutations on the receptor desensitization and internalization will be examined. Also, the role of receptor phosphorylation on the superactivation of adenylyl cyclase will be elucidated by identifying the cellular proteins that are recruited to the vicinity of receptor complexes. The role of these proteins in expressing the superactivation of the adenylyl cyclase activity will be examined. These studies and others will lead to eventual understanding of cellular control of -opioid receptor activities and subsequently, the understanding of morphine chronic actions.